In the nearly 20 years since the groundbreaking sequencing of the human genome, the potential for precisiongenetic medicine has still not been realized. Advances in sequencing have vastly increased our ability to screenpatients (including parents and fetuses) for genetic abnormalities, dramatically expanding identification ofhereditary diseases in the prenatal period. This opens the door for fetal molecular therapies to address criticalgenetic conditions in utero, preventing preterm pregnancy termination, newborn death, or irreversible tissuedamage resulting in life-long disabilities. The discovery of CRISPR systems and their remarkable ability toperform precision gene editing quickly showed possibilities for clinical applications to treat genetic disorders.However, clinical application of CRISPR is limited due to the introduction of mutations and DNA restructuring atunintended off-target sites within the genome, which can lead to toxicity and cancer. Nowhere is this deficiencymore acute than in the application of CRISPR for in utero gene editing, where uncontrolled editing side effectscould affect the patient for the entirety of their lives. Controlling CRISPR gene editing is critical to ensure thesafety and efficacy of in utero gene therapies. Acrigen Biosciences is commercializing technology to bringsafe in vivo CRISPR-based gene editing therapies to the clinic. Acrigen utilizes anti-CRISPR (Acr) proteins asrobust inhibitors of Cas nuclease, providing an off-switch for gene editing and preventing off-target effects. TheMacKenzie lab at UCSF specializes in fetal surgery and applying molecular therapies to correct neonatal geneticdiseases. The combination of controlled CRISPR gene editing with precise in utero delivery will allow us toaddress previously untreatable genetic disorders, including hematopoietic disorders like alpha thalassemia andneurologic diseases such as neuropathic Gaucher disease. We propose to develop a safe and effective in uteroCRISPR gene editing delivery system targeting hematopoietic stem cells and neurons in a fetal mouse model.This Phase I STTR project has three aims. Aim 1: Design SaCas9 guides targeting a mouse reporter gene.Milestone 1: Select guides showing >70% editing in reporter mouse cell line. Aim 2: Construct a single vector(Cas9-sgRNA-Acr) delivery system for controlled in utero editing. Milestone 2: Demonstrate maintained on-targetactivity and >90% reduction of off-target activity with an AAV compatible single vector system under Acr control.Aim 3: Demonstrate safe and effective editing of a reporter mouse model in utero. Milestone 3: Show >50% on-target editing and <5% off-target editing with AAV6 targeting hematopoietic stem cells and AAV9 targetingneurons. Narrative
Combining precision fetal surgery with controlled gene editing will enable in utero correction of severe genetic
disorders. Acrigen Biosciences, Inc and the MacKenzie group at UCSF are developing technology to control
CRISPR gene editing and deliver precision genetic medicines directly to the developing fetus, enabling safe
and effective therapies for neonatal genetic diseases. Genetic defect ; genome mutation ; Persons ; Neonatal Mortality ; neonatal mortalities ; newborn death ; newborn mortality ; nervous system disorder ; Nervous System Diseases ; Neurologic Disorders ; Neurological Disorders ; neurological disease ; Neurons ; Nerve Cells ; Nerve Unit ; Neural Cell ; Neurocyte ; neuronal ; Parents ; Patients ; Phenotype ; Pregnancy ; Gestation ; Proteins ; Research ; Safety ; Technology ; Drug or chemical Tissue Distribution ; Tissue Distribution ; Tissues ; Body Tissues ; Genetic Variation ; Genetic Diversity ; Work ; Guide RNA ; gRNA ; base ; Organ ; Site ; Acute ; Clinical ; Phase ; Reporter Genes ; Ensure ; Evaluation ; Hepatocyte ; Hepatic Cells ; Hepatic Parenchymal Cell ; Liver Cells ; Hematopoietic ; hemopoietic ; disability ; Childhood ; pediatric ; Molecular Abnormality ; Chromosomal, Gene, or Protein Abnormality ; Cytogenetic or Molecular Genetic Abnormality ; Genetic Abnormality ; molecular aberrations ; Therapeutic ; Genetic ; Reporter ; Life ; Clinic ; neuropathic ; Neuropathy ; Source ; System ; fetal ; Surgeon ; nuclease ; Animal Models and Related Studies ; model of animal ; model organism ; Animal Model ; Toxicities ; Toxic effect ; unborn ; prenatal ; Reporting ; fetal surgery ; fetus surgery ; preventing ; prevent ; Address ; Mouse Cell Line ; in vivo ; Fetal Liver ; Genetic Medicine ; Small Business Technology Transfer Research ; STTR ; Validation ; Molecular ; Text ; Development ; developmental ; in utero ; vector ; design ; designing ; Outcome ; Neonatal ; Coupling ; innovation ; innovate ; innovative ; Oncogenic ; Cell model ; Cellular model ; clinical application ; clinical applicability ; human disease ; mouse model ; murine model ; effective therapy ; effective treatment ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR ; CRISPR/Cas system ; precision genetics ; personalized genetics ; human genome sequencing ; CRISPR/Cas technology ; CRISPR method ; CRISPR methodology ; CRISPR technique ; CRISPR technology ; CRISPR-CAS-9 ; CRISPR-based method ; CRISPR-based technique ; CRISPR-based technology ; CRISPR-based tool ; CRISPR/Cas method ; CRISPR/Cas9 ; CRISPR/Cas9 technology ; Cas nuclease technology ; Clustered Regularly Interspaced Short Palindromic Repeats method ; Clustered Regularly Interspaced Short Palindromic Repeats methodology ; Clustered Regularly Interspaced Short Palindromic Repeats technique ; Clustered Regularly Interspaced Short Palindromic Repeats technology ; patient screening ; Genetic Diseases ; genetic condition ; genetic disorder ; therapeutic genome editing ; gene-editing therapy ; genome editing based therapy ; genome editing therapy ; genome editing treatment ; genome editing-based therapeutics ; therapeutic editing ; side effect ; blood-brain barrier permeabilization ; BBB permeabilization ; BBB permeable ; blood-brain barrier permeable ; bloodbrain barrier permeabilization ; bloodbrain barrier permeable ; off-target site ; Termination of pregnancy ; Affect ; alpha-Thalassemia ; Hemoglobin H Disease ; secondary leukemia ; treatment associated acute myelogenous leukemia ; α-thalassemia ; inhibitor/antagonist ; inhibitor ; Biological Sciences ; Biologic Sciences ; Bioscience ; Life Sciences ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Disease ; Disorder ; DNA ; Deoxyribonucleic Acid ; Fetal Development ; Developing fetus ; Fetus ; Fluorescence ; Gaucher Disease ; Gauchers Disease ; gene therapy ; DNA Therapy ; Gene Transfer Clinical ; Genetic Intervention ; gene-based therapy ; genetic therapy ; genomic therapy ; Genes ; Switch Genes ; Genome ; Goals ; Government ; Hematopoietic stem cells ; Blood Precursor Cell ; Hematopoietic Progenitor Cells ; blood stem cell ; hematopoietic progenitor ; hematopoietic stem progenitor cell ; hemopoietic progenitor ; hemopoietic stem cell ; Hereditary Disease ; Inborn Genetic Diseases ; Inherited disorder ; hereditary disorder ; heritable disorder ; inborn error ; inherited diseases ; inherited genetic disease ; inherited genetic disorder ; Human ; Modern Man ; Lead ; Pb element ; heavy metal Pb ; heavy metal lead ; Biological Models ; Biologic Models ; Model System ; Mus ; Mice ; Mice Mammals ; Murine ; Mutation ; Genetic Alteration ; Genetic Change ;
